Pharmaceutical composition comprising an extract of Pseudolysimachion longifolium and the catalpol derivatives isolated therefrom having antiinflammatory, antiallergic and antiasthmatic activity

ABSTRACT

The present invention relates to a composition comprising an extract of  Pseudolysimachion  genus plant, and the catalpol derivatives isolated therefrom having anti-inflammatory, anti-allergic and anti-asthmatic activity. The extract of  Pseudolysimachion  genus plant and the catalpol derivatives isolated therefrom shows potent suppressing effect on elevated IgE, IL-4 and IL-13 levels and eosinophilia in the plasma and BALF, and mucus overproduction in the lung tissues in an OVA-induced asthmatic mouse model. Therefore, it can be used as the therapeutics or functional health food for treating and preventing inflammatory, allergic and asthmatic disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/916,216, filed Nov. 30, 2007, now U.S. Pat. No. 8,168,235 which is a371 of PCT/KR2006/002092, filed May 30, 2006, which claims the benefitof Korean Patent Application Nos. 10-2005-0045756, filed May 30, 2005,10-2005-0045755, filed May 30, 2005, 10-2006-0048319, filed May 29,2006, and 10-2006-0048104, filed May 29, 2006, the contents of each ofwhich are incorporated herein by reference its entirety.

TECHNICAL FIELD

The present invention relates to a composition comprising an extract ofPseudolysimachion longifolium and the catalpol derivatives isolatedtherefrom having anti-inflammatory, anti-allergic and anti-asthmaticactivity.

BACKGROUND ART

Asthma has been regarded as a complex syndrome occurring in the airways,which shows various disorders such as airflow obstruction, acute orchronic inflammation, airway hyper-responsiveness (AHR) and structuralremodeling (Kumar R. K. Pharmacol. Ther., 91, pp 93-104, 2001).

Allergic inflammation occurring in the airways has been reported to playa critical role in asthma development and the number of patientssuffering from allergic asthma has been increased to about 10% of thepopulation in the world recently. It has been reported that the numberhas been reached to seventeen million in America and the market scale ofthe medication for allergic asthma has been enlarged to 640 billion $ inAmerica till now.

Asthma can be classified into two types, i.e., extrinsic asthma andintrinsic asthma. Extrinsic asthma caused by the exposure of antigensuch as house dust mite Dermatophagoides as a main antigen, pollen,epithelium of animal, fungi etc shows positive reaction in skin test orbronchial provocation test against the antigen, and generally occurs inyounger people. Intrinsic asthma caused by upper respiratory infection,exercise, emotional instability, cold weather, the change of humidityoccurs in adult patients.

According to the aspect of pathophysiology, asthma has been recognizedas a chronic inflammation occurred by following procedure; Inflammatorycells are proliferated, differentiated, and activated caused bycytokines reproducing in T-helper 2 immune cells and is moved to air wayor neighboring tissue thereof. The activated inflammatory cells such asneutrophil, mast cell etc release a variety of inflammatory mediators,such as cytokines, chemokines, signaling molecules, adhesion moleculesand growth factors and the structural cells in airways are involved invarious stages of asthma (Elias J A et al., J Clin Invest., 111, pp291-7, 2003). In numerous studies using knockout mice models andclinical research, the critical observations in asthma could fall intoseveral characteristic parameters, such as immune responses,eosinophilia, AHR and structural remodeling (Moffatt J D. Pharmacol Ther107, pp 343-57, 2005; Spina D et al., Trends Pharmacol Sci, 23, pp311-5, 2002). Each of the parameters seems not to have directcorrelations with one another; however, IgE-mediated immune response andeosinophilia are prominent symptoms in the airways of allergic asthma(Bochner B. S. et al., Annu. Rev. Immunol., 12, pp 295-335, 1994;Bousquet J et al., N. Engl. J. Med., 323, pp 1033-9, 1990), and theproduced cytokines such as IL-4, IL-5 and IL-13 in the allergic processalso play an important role in AHR development and airway remodeling(Riffo-Vasquez Y et al., Pharmacol. Ther., 94, pp 185-211, 2002).Indeed, asthma is a result of orchestrated inflammatory events, many ofwhich involve specific inhibitors acting on the pathway of asthma, forexample, histamine H1 antagonists, thromboxane antagonists,platelet-activating-factor antagonists, cyclooxygenase inhibitors,nitrogen monooxygenase inhibitors and prostaglandin inhibitors, havebeen tried but have failed in clinical trials (Moffatt J. D., Pharmacol.Ther., 107, pp 343-57, 2005). In contrast, glucocorticoids, whichsuppress the progenitor levels of inflammatory cells to baseline bywidespread inhibition of cytokine synthesis and cytokine mediatedimmune-cell survival, has been used to manage the symptoms of asthmaticpatients over a period of 30 years as far (Baatjes A. J. et al.,Pharmacol, Ther., 95, pp 63-72, 2002). These reports suggest that thetherapeutic approach for asthma management should focus on restoring thebalance of asthmatic parameters rather than searching for potentinhibitors of specific pathways of the asthmatic process.

Pseudolysimachion longifolium belonged to Pseudolysimachion genus, is aperennial herb distributed in Korea, China, Russia and Europe. Numerousspecies of same genus for example, Pseudolysimachion ovutum,Pseudolysimachion kiusianum, Pseudolysimachion kiusianum vardiamanticum, Pseudolysimachion kiusianum var villosum, Pseudolysimachiondahuricum, Pseudolysimachion pyrethrinum, Pseudolysimachionlinarifolium, Pseudolysimachion linarifolium var. villosulum,Pseudolysimachion rotundum var. subintegrum, Pseudolysimachion rotundumvar. coreanum, Pseudolysimachion insulare, and Pseudolysimachionundulata have been reported and the plants contains mannitol,6-hydroxyluteolin as a main ingredient (Chung B S and Shin M K,HyangyakDaeSaJeon, Youngrimsa, pp 913-914, 1998).

However, there has been not reported or disclosed about the suppressiveeffect on inflammatory, allergic and asthmatic disease of the extractfrom P. longifolium and the catalpol derivatives isolated therefrom inany of above cited literatures, the disclosures of which areincorporated herein by reference.

Accordingly, the present inventors have discovered that the extract ofP. longifolium and the catalpol derivatives isolated therefrom show thesuppressive effect on asthmatic parameters, such as IgE level, cytokinerelease, and eosinophilia, AR and mucus hypersecretion inOVA-sensitized/challenged mouse model and finally completed the presentinvention.

SUMMARY OF THE INVENTION

The present invention provides a pharmaceutical composition and a healthfood comprising an extract or catalpol derivatives isolated from P.longifolium as an active ingredient in an effective amount to treat andprevent inflammatory, allergic and asthmatic disease.

The present invention also provides a use of an extract of P.longifolium and the catalpol derivatives isolated therefrom showinganti-inflammatory, anti-allergic and anti-asthmatic activity.

The present invention also provides a method of treating or preventinginflammatory, allergic and asthmatic disease in a mammal comprisingadministering to said mammal an effective amount of an extract of P.longifolium and the catalpol derivatives isolated therefrom, togetherwith a pharmaceutically acceptable carrier thereof.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, it is an object of the present invention to provide acomposition comprising a crude extract or organic solvent solubleextract of Pseudolysimachion genus plant, as an active ingredient forthe treatment and prevention of inflammatory, allergic and asthmaticdisease.

The term “crude extract” disclosed herein comprises the extract preparedby extracting plant material with water, lower alcohols such asmethanol, ethanol, preferably methanol and the like, or the mixturesthereof.

The term “organic solvent soluble extract” disclosed herein can beprepared by extracting the above described crude extract with organicsolvent, for example, butanol, acetone, ethyl acetate, chloroform ordichloromethane, preferably butanol. The term “Pseudolysimachion genus”disclosed herein comprises P. longifolium, P. ovtum, P. kiusianum, P.kiusianum var. diamanticum, P. kiusianum var. villosum, P. dahuricum, P.pyrethrinum, P, linarifolium, P. linarifolium var. villosulum, P.rotundum var. subintegrum, P. rotundum var. coreanum, P. insulare and P.undulate.

The present invention provides a pharmaceutical composition comprisingcatalpol derivatives represented by following chemical formula (I), or apharmaceutically acceptable salt thereof as an active ingredient in aneffective amount to treat and prevent inflammatory, allergic and asthmadisease:

wherein,

R is independently at least one groups selected from a hydrogen atom,benzoyl or cinnamoyl group substituted with C₁-C₃ lower alkyl group orC₁-C₃ lower alkoxy group.

In the above formula (I), preferably, R group comprises3,4-dihydroxybenzoyl, 4-hydroxy-3-methozybenzoyl,3-hydroxy-4-methozybenzoyl, 4-hydroxybenzoyl, 3,4-dimethoxybenzoyl,3,4-dihydroxycinnamoyl and 3-hydroxy-4-methoxycinnamoyl.

The catalpol derivatives of the present invention can be isolated fromP. longifolium or synthesized by general procedure well known in the art(Herbert O. house., Modern Synthetic Reactions, 2^(nd) Ed., TheBenjamin/Cummings Publishing Co., 1972).

In accordance with another aspect of the present invention, there isalso provided a use of crude extract or organic solvent soluble extractof Pseudolysimachion genus plant, or the catalpol derivatives isolatedtherefrom for manufacture of medicines employed for treating orpreventing inflammatory, allergic and asthmatic disease.

In accordance with another aspect of the present invention, there isalso provided a method of treating or preventing inflammatory, allergicand asthmatic disease in mammals, wherein the method comprisesadministering a therapeutically effective amount of crude extract ororganic solvent soluble extract of Pseudolysimachion genus plant, or thecatalpol derivatives isolated therefrom into the mammal suffering withinflammatory, allergic and asthmatic disease.

An inventive extract isolated from Pseudolysimachion genus plant, andthe catalpol derivatives isolated therefrom may be prepared inaccordance with the following preferred embodiment.

Hereinafter, the present invention is described in detail.

For the present invention, for example, the dried leave of P.longifolium is cut into small pieces and the piece was mixed with 2 to20-fold, preferably, 5 to 10-fold volume of polar solvent, for example,water, C₁-C₄ lower alcohol such as methanol, ethanol, butanol, or themixtures thereof, preferably methanol; and was heated at the temperatureranging from 20 to 100° C., preferably from 20 to 50° C., for the periodranging 10 to 48 hours, preferably 20 to 30 hours, by reflux extractionwith hot water, cold water extraction, ultra-sonication or conventionalextraction, preferably by cold water extraction; the residue wasfiltered and then the filtrate is dried to obtain polar solvent solubleextract thereof.

In the above crude extract prepared by above step, is suspended inwater, and then is mixed with 1 to 100-fold, preferably, 1 to 5-foldvolume of organic solvent butanol, acetone, ethyl acetate, chloroform ordichloromethane, preferably butanol to obtain organic solvent solubleextract of the present invention.

The above organic solvent soluble extract is further subjected to silicagel column chromatography filled with silicagel eluting with mixturesolvent of chloroform:methanol with increasing the polarity by changingthe mixed ratio (methanol 0-100%, step gradient) to obtain severalfractions. Among the fractions, the 3^(rd) fraction is further subjectedto repeated silica gel column chromatography using a normal phase silicacolumn (methanol 10-50% step gradient) to obtain the catalpolderivatives of the present invention. The structure was confirmed byNMR, EI-MS and optical rotation with those reported previously(Afifi-Yazar F Ü et al., Helv Chim Acta, 63, pp 1905-7, 1980) and thepurity of the catalpol derivatives was analyzed as more than 99.5% byHPLC system.

In accordance with another aspect of the present invention, there isprovided a pharmaceutical composition comprising a crude extract andorganic solvent soluble extract of P. longifolium or the catalpolderivatives isolated therefrom prepared by the above describepreparation method for the treatment and prevention of inflammatory,allergic and asthmatic disease as active ingredients.

In accordance with another aspect of the present invention, there isalso provided a use of comprising a crude extract and organic solventsoluble extract of P. longifolium or the catalpol derivatives isolatedtherefrom prepared by the above describe preparation method formanufacture of medicines employed for treating or preventinginflammatory, allergic and asthmatic disease.

In accordance with another aspect of the present invention, there isalso provided a method of treating or preventing inflammatory, allergicand asthmatic disease, wherein the method comprises administering atherapeutically effective amount of comprising a crude extract andorganic solvent soluble extract of P. longifolium or the catalpolderivatives isolated therefrom prepared by the above describepreparation method.

The inventive compound represented by general formula (I) can betransformed into their pharmaceutically acceptable salt and solvates bythe conventional method well known in the art. For the salts,acid-addition salt thereof formed by a pharmaceutically acceptable freeacid thereof is useful and can be prepared by the conventional method.For example, after dissolving the compound in the excess amount of acidsolution, the salts are precipitated by the water-miscible organicsolvent such as methanol, ethanol, acetone or acetonitrile to prepareacid addition salt thereof and further the mixture of equivalent amountof compound and diluted acid with water or alcohol such as glycolmonomethylether, can be heated and subsequently dried by evaporation orfiltrated under reduced pressure to obtain dried salt form thereof.

As a free acid of above-described method, organic acid or inorganic acidcan be used. For example, organic acid such as methansulfonic acid,p-toluensulfonic acid, acetic acid, trifluoroacetic acid, citric acid,maleic acid, succinic acid, oxalic acid, benzoic acid, lactic acid,glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaricacid, glucuronic acid, aspartic acid, ascorbic acid, carbonylic acid,vanillic acid, hydroiodic acid and the like, and inorganic acid such ashydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaricacid and the like can be used herein.

Further, the pharmaceutically acceptable metal salt form of inventivecompounds may be prepared by using base. The alkali metal oralkali-earth metal salt thereof can be prepared by the conventionalmethod, for example, after dissolving the compound in the excess amountof alkali metal hydroxide or alkali-earth metal hydroxide solution, theinsoluble salts are filtered and remaining filtrate is subjected toevaporation and drying to obtain the metal salt thereof. As a metal saltof the present invention, sodium, potassium or calcium salt arepharmaceutically suitable and the corresponding silver salt can beprepared by reacting alkali metal salt or alkali-earth metal salt withsuitable silver salt such as silver nitrate.

The pharmaceutically acceptable salt of the present compound compriseall the acidic or basic salt which may be present at the compounds, ifit does not indicated specifically herein. For example, thepharmaceutically acceptable salt of the present invention comprise thesalt of hydroxyl group such as the sodium, calcium and potassium saltthereof; the salt of amino group such as the hydrogen bromide salt,sulfuric acid salt, hydrogen sulfuric acid salt, phosphate salt,hydrogen phosphate salt, dihydrophosphate salt, acetate salt, succinatesalt, citrate salt, tartarate salt, lactate salt, mandelate salt,methanesulfonate(mesylate) salt and p-toluenesulfonate (tosylate) saltetc, which can be prepared by the conventional method well known in theart.

The inventive composition for treating and preventing inflammatory,allergic and asthmatic disease may comprises the above describedextracts or compounds as 0.1-50% by weight based on the total weight ofthe composition.

The composition according to the present invention can be provided as apharmaceutical composition containing pharmaceutically acceptablecarriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose,sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acaciarubber, alginate, gelatin, calcium phosphate, calcium silicate,cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxy benzoate, talc, magnesium stearate and mineraloil. The formulations may additionally include fillers,anti-agglutinating agents, lubricating agents, wetting agents, flavoringagents, emulsifiers, preservatives and the like. The compositions of theinvention may be formulated so as to provide quick, sustained or delayedrelease of the active ingredient after their administration to a patientby employing any of the procedures well known in the art.

For example, the compositions of the present invention can be dissolvedin oils, propylene glycol or other solvents that are commonly used toproduce an injection. Suitable examples of the carriers includephysiological saline, polyethylene glycol, ethanol, vegetable oils,isopropyl myristate, etc., but are not limited to them. For topicaladministration, the extract of the present invention can be formulatedin the form of ointments and creams.

Pharmaceutical formulations containing present composition may beprepared in any form, such as oral dosage form (powder, tablet, capsule,soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet,granule), or topical preparation (cream, ointment, lotion, gel, balm,patch, paste, spray solution, aerosol and the like), or injectablepreparation (solution, suspension, emulsion).

The composition of the present invention in pharmaceutical dosage formsmay be used in the form of their pharmaceutically acceptable salts, andalso may be used alone or in appropriate association, as well as incombination with other pharmaceutically active compounds.

The desirable dose of the inventive extract or compound varies dependingon the condition and the weight of the subject, severity, drug form,route and period of administration, and may be chosen by those skilledin the art. However, in order to obtain desirable effects, it isgenerally recommended to administer at the amount ranging from 0.0001 to100 mg/kg, preferably, 0.001 to 10 mg/kg by weight/day of the inventiveextract of the present invention. The dose may be administered in singleor divided into several times per day.

The pharmaceutical composition of present invention can be administeredto a subject animal such as mammals (rat, mouse, domestic animals orhuman) via various routes. All modes of administration are contemplated,for example, administration can be made orally, rectally or byintravenous, intramuscular, subcutaneous, intracutaneous, intrathecal,epidural or intracerebroventricular injection.

It is the other object of the present invention to provide a functionalhealth food comprising the extract or compounds isolated from P.longifolium together with a sitologically acceptable additive for theprevention and alleviation of inflammatory, allergic and asthmaticdisease.

To develop for functional health food, examples of addable foodcomprising the above extracts or compounds of the present invention arevarious food, beverage, gum, vitamin complex, health improving food andthe like, and can be used as powder, granule, tablet, chewing tablet,capsule or beverage etc.

The above described composition therein can be added to food, additiveor beverage, wherein, the amount of the above described extract orcompound in food or beverage may generally range from about 0.01 to 80w/w %, preferably 0.01 to 15 w/w % of total weight of food for thehealth food composition and 0.02 to 5 g, preferably 0.3 to 1 g on theratio of 100 mL of the health beverage composition.

Providing that the health beverage composition of present inventioncontains the above described extract or compound as an essentialcomponent in the indicated ratio, there is no particular limitation onthe other liquid component, wherein the other component can be variousdeodorant or natural carbohydrate etc such as conventional beverage.Examples of aforementioned natural carbohydrate are monosaccharide suchas glucose, fructose etc; disaccharide such as maltose, sucrose etc;conventional sugar such as dextrin, cyclodextrin; and sugar alcohol suchas xylitol, and erythritol etc. As the other deodorant thanaforementioned ones, natural deodorant such as taumatin, stevia extractsuch as levaudioside A, glycyrrhizin et al., and synthetic deodorantsuch as saccharin, aspartam et al., may be useful favorably. The amountof above described natural carbohydrate is generally ranges from about 1to 20 g, preferably 5 to 12 g in the ratio of 100 mL of present beveragecomposition.

The other components than aforementioned composition are variousnutrients, a vitamin, a mineral or an electrolyte, synthetic flavoringagent, a coloring agent and improving agent in case of cheese chocolateet al., pectic acid and the salt thereof, alginic acid and the saltthereof, organic acid, protective colloidal adhesive, pH controllingagent, stabilizer, a preservative, glycerin, alcohol, carbonizing agentused in carbonate beverage et al. The other component thanaforementioned ones may be fruit juice for preparing natural fruitjuice, fruit juice beverage and vegetable beverage, wherein thecomponent can be used independently or in combination. The ratio of thecomponents is not so important but is generally range from about 0 to 20w/w % per 100 w/w % present composition. Examples of addable foodcomprising aforementioned extract therein are various food, beverage,gum, vitamin complex, health improving food and the like.

Inventive extract of the present invention has no toxicity and adverseeffect therefore they can be used with safe.

The present invention is more specifically explained by the followingexamples. However, it should be understood that the present invention isnot limited to these examples in any manner.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which;

FIG. 1 presents the effects of verproside isolated from P. longifoliumon airway hyperresponsiveness (AHR) in the ovalbumin (OVA) andmethacholine challenged cells compared to montelukast (ML).

FIG. 2 presents the effects of verproside isolated from P. longfolium onon OVA-Induced Eosinophilia in BALF in the ovalbumin (OVA) challengedmice compared to montelukast (ML).

FIG. 3 presents the effects of verproside isolated from P. longifoliumand P. longifoliumextract on eosinophilia by the lung tissues stainedwith hematoxylin (I: Normal Control; II: OVA-challenged mice; III: OVAchallenge +verproside treated mice; IV: OVA challenge +Montelukasttreated; V: OVA challenged +P. longifolium extract-treated mice).

FIG. 4 presents the effects of verproside isolated from P. longifoliumon eosinophilia by the lung tissues stained in Periodic Acid Schiff(PAS) staining (I: Normal Control; II: OVA-challenged mice; III: OVAchallenge +verproside treated mice; IV: OVA challenge +Montelukasttreated).

FIG. 5 presents the effects of verproside isolated from P. longfolium onmucus hypersecretion in the bronchial airways (NC: Normal Control; OVA:OVA-challenged mice; verproside: OVA challenge +verproside treated mice;ML: OVA challenged +Montelukast treated).

FIG. 6 presents the effects of verproside isolated from P. longifoliumon IgE levels and cytokines, IL-4 and IL-3, concentrations in BALF ofthe asthmatic model compared to montelukast (NC: Normal Control; OVA:OVA-challenged mice; Verproside: OVA challenge +verproside treated mice;ML: OVA cahllenged +Montelukast treated).

BEST MODE FOR CARRYING OUT THE INVENTION

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the compositions, use andpreparations of the present invention without departing from the spiritor scope of the invention.

The present invention is more specifically explained by the followingexamples. However, it should be understood that the present invention isnot limited to these examples in any manner.

EXAMPLES

The following Reference Example, Examples and Experimental Examples areintended to further illustrate the present invention without limitingits scope.

Example 1 Preparation of the Crude Extract of P. Longifolium

7.9 kg of dried P. longifolium cut into small pieces, mixed with 50 L ofmethanol and the mixture was stirred at room temperature for 24 hours,extracted by cold water three times. The extract was filtered withfilter paper to remove the debris. The filtrate was pooled andconcentrated by rotary evaporator 55-65° C. under reduced pressure anddried with freezing dryer to obtain 950.5 g of dried crude extract of P.longifolium.

Example 2 Preparation of Polar Solvent and Non-Polar Solvent SolubleExtract

2-1. Preparation of Ethyl Acetate Soluble Fraction

10 L of distilled water was added to 425 g of the crude extract obtainedin Example 1. 10 L of ethyl acetate was added thereto in separatoryfunnel and shaken vigorously to divide into ethyl acetate soluble layerand water soluble layer.

Above ethyl acetate soluble layer was concentrated by rotary evaporator,dried with freeze dryer to obtain ethyl acetate soluble extract.

2-2. Preparation of Butanol/Water Soluble Fraction

Water soluble layer was fractionated by mixing with 10 L of butanol andfinally, 144.0 g of n-butanol soluble extract and water soluble extractwere obtained to use as a sample in the following experiments.

Example 3 Preparation of Catalpol Derivatives from the Extract of P.Longifolium

3-1. Preparation of Verproside(6-O-3,4-Dihydroxybenzoyl Catalpol)

144.0 g of n-butanol soluble fraction was subjected to a silica gelcolumn chromatography (70-230 mesh, 8.5×65 cm) and eluted with achloroform-methanol mixture (methanol 0-100%, step gradient) to obtainfive fractions. 29.1 g of fraction 2 (between chloroform-methanol7/3-6/4, v/v) was subjected to repeated column chromatography using anormal phase silica column chromatography (silica gel, 230-400 mesh,6.0×60 cm, chloroform-methanol mixture, methanol 10-50% step gradient).The fractions 2-4 was performed to recrystallization in methanol toobtain 14.2 g of verproside, i.e., 6-O-3,4-Dihydroxybenzoyl catalpol.The structure was confirmed by NMR (¹H, ¹³C, DEPT, HMQC, HMBC), EI-MSand optical rotation with those reported previously (Afifi-Yazar F Ü etal., Helv Chim Acta, 63, pp 1905-7, 1980) and the purity of verprosidewas analyzed as more than 99.5% by HPLC system (Shimadzu SCL-10A withSPD-M 10A vp PDA detector, column; Phenomenex Synergi 4 um Fusion RP-80,4.6×150 mm, elution: MeOH/DW, 35/65, v/v, 0.8 ml/min).

6-O-3,4-Dihydroxybenzoyl catalpol(verproside)

¹H NMR (400 MHz, DMSO-d₆) δ: 2.47 (1H, dd, J=8.0, 9.2 Hz, H-9), 2.59(1H, dddd, J=1.6, 4.0, 8.0, 8.0, H-5), 3.00 (1H, m, H-G4), 3.05 (1H, m,H-G2), 3.14 (1H, m, H-G5), 3.18 (1H, m, H-G3), 3.42, 3.71 (2H, m, H-G6).3.67 (1H, s, H-7), 3.71, 3.91 (2H, d, J=13.2 Hz, each, H-10), 4.61 (1H,d, J=7.6 Hz, H-G1), 4.94 (1H, dd, J=4.0, 6.0 Hz, H-4), 5.03 (1H, d,J=8.0 Hz, H-6), 5.09 (1H, d, J=9.2 Hz, H-1), 6.41 (1H, dd, J=1.6. 6.0Hz, H-3), 6.82 (1H, d, J=8.0 Hz, H-5′), 7.35 (1H, dd, J=2.0, 8.0 Hz,H-6′), 7.39 (1H, d, J=2.0 Hz, H-2′).

¹³C-NMR (100 MHz, DMSO-d₆) δ: 93.0 (C-1), 141.1 (C-3), 101.8 (C-4), 35.2(C-5), 79.5 (C-6), 58.2 (C-7), 65.8 (C-8), 41.8 (C-9), 120.0 (C-1′),116.4 (C-2′), 145.1 (C-3′), 150.8 (C-4′), 115.4 (C-5′), 122.6 (C-6′),165.6 (C-7′), 97.9 (C-G1), 73.4 (C-G2), 76.4 (C-G3), 70.3 (C-G4), 77.5(C-G5), 61.4 (C-G6).

3-2. Preparation of Isovanillyl Catalpol from the Extract of P.Longifolium

17.3 g of fraction 3 was subjected to column chromatography using anormal phase silica column (silica gel, 230-400 mesh, 6.0×60 cm,chloroform-methanol mixture, methanol 10-50% step gradient). 8.5 g offraction 3-3 was performed to recrystallization in methanol to obtain7.2 g of isovanillyl catalpol, i.e., 6-O-3-hydroxy-4-methoxybenzolycatalpol.

6-O-3-hydroxy-4-methoxybenzoly catalpol(isovanillyl catalpol)

¹H-NMR (400 MHz, DMSO-d₆) δ: 2.47 (1H, m, H-9), 2.55 (1H, m H-5), 3.00(1H, m, H-G4), 3.05 (1H, m, H-G2), 3.14 (1H, m, H-G5), 3.18 (1H, m,H-G3), 3.43, 3.70 (2H, m, H-G6), 3.70 (1H, br s, H-7), 3.72, 3.92 (2H,d, J=13.2, each, H-10), 4.62 (1H, d, J=8.0 Hz, H-G1), 4.95 (1H, dd,J=4.4, 6.0 Hz, H-4), 5.06 (1H, d, J=8.0 Hz, H-6), 5.11 (1H, d, J=9.2 Hz,H-1), 6.42 (1H, d, J=6.0 Hz, H-3), 7.04 (1H, d, J=8.4 Hz, H-5′), 7.42(1H, br s, H-2′), 7.48 (1H, d, J=8.4 Hz, H-6′), 3.84 (3H, s, 4′-O—CH₃).

¹³C-NMR (100 MHz, DMSO-d₆) δ: 93.0 (C-1), 141.0 (C-3), 101.6 (C-4), 35.2(C-5), 79.7 (C-6), 58.2 (C-7), 65.8 (C-8), 41.8 (C-9), 58.4 (C-10),121.7 (C-1′), 115.7 (C-2′), 146.3 (C-3′), 152.1 (C-4′), 111.4 (C-5′),121.3 (C-6′), 165.3 (C-7′), 97.8 (C-G1), 73.4 (C-G2), 76.4 (C-G3), 70.3(C-G4), 77.4 (C-G5), 61.4 (C-G6), 55.7 (4′-OCH₃).

3-3. Preparation of PicrosideII and Verminoside from the Extract of P.Longifolium

1.5 g of fraction 3-5 was subjected to reversed phase silica gel column(RP-18, YMC Gel ODS-A, 6.0×60 cm, methanol/water, 1/4, v/v), subjectedto sepadex LH-20 column chromatography(methanol/water, 85/15, v/v) toobtain 101.0 mg of picrosideII, i.e., 6-O-4-hydroxy-3-methozybenzoyl and30.0 mg of verminoside, i.e., 6-O-3,4-dihydroxycinnamoyl catalpol.

6-O-4-hydroxy-3-methozybenzoyl (picrosideII)

¹H-NMR (400 MHz, DMSO-d₆) δ: 2.47 (1H, dd, J=8.0, 9.6 Hz, H-9), 2.58(1H, dddd, J=1.2, 6.0, 8.0, 8.4 Hz, H-5), 3.00 (1H, m, H-G4), 3.05 (1H,m, H-G2), 3.14 (1H, m, H-G5), 3.18 (1H, m, H-G3), 3.42, 3.71 (2H, m,H-G6), 3.67 (1H, br s, H-7), 3.72, 3.92 (2H, d, J=13.2, each, H-10),4.62 (1H, d, J=7.6 Hz, H-G1), 4.99 (1H, dd, J=4.4, 6.0 Hz, H-4), 5.06(1H, d, J=8.4 Hz, H-6), 5.11 (1H, d, J=9.6 Hz, H-1), 6.42 (1H, dd,J=1.2. 6.0 Hz, H-3), 6.89 (1H, d, J=8.4 Hz, H-5′), 7.46 (1H, d, J=2.0Hz, H-2′), 7.52 (1H, dd, J=2.0, 8.4 Hz, H-6′), 3.83 (3H, s, 3′-O—CH₃).

¹³C-NMR (100 MHz, DMSO-d₆) δ: 93.0 (C-1), 141.1 (C-3), 101.8 (C-4), 35.2(C-5), 79.7 (C-6), 58.2 (C-7), 65.8 (C-8), 41.8 (C-9), 58.5 (C-10),120.0 (C-1′), 112.7 (C-2′), 147.5 (C-3′), 152.0 (C-4′), 115.3 (C-5′),123.8 (C-6′), 165.6 (C-7′), 97.9 (C-G1), 73.4 (C-G2), 76.4 (C-G3), 70.3(C-G4), 77.5 (C-G5), 61.4 (C-G6), 55.7 (3′-OCH₃).

6-O-3,4-dihydroxycinnamoyl catalpol(verminoside)

¹H-NMR (400 MHz, DMSO-d₆) δ: 2.43 (1H, m, H-9), 2.45 (1H, m, H-5), 3.01(1H, m, H-G4), 3.05 (1H, m, H-G2), 3.14 (1H, m, H-G5), 3.18 (1H, m,H-G3), 3.42, 3.70 (2H, m, H-G6), 3.64 (1H, br s, H-7), 3.71, 3.90 (2H,d, J=13.2 Hz, each, H-10), 4.61 (1H, d, J=8.4 Hz, H-G1), 4.94 (1H, dd,J=4.0, 5.6 Hz, H-4), 4.99 (1H, d, J=7.2 Hz, H-6), 5.08 (1H, d, J=9.2 Hz,H-1), 6.42 (1H, d, J=5.6 Hz, H-3), 6.77 (1H, d, J=8.0 Hz, H-5′), 7.08(1H, d, J=1.6 Hz, H-2′), 7.05 (1H, dd, J=1.6, 8.0 Hz, H-6′).

¹³C-NMR (100 MHz, DMSO-d₆) δ: 92.9 (C-1), 141.1 (C-3), 101.7 (C-4), 35.1(C-5), 79.2 (C-6), 58.2 (C-7), 65.7 (C-8), 41.8 (C-9), 58.5 (C-10),125.4 (C-1′), 115.8 (C-2′), 146.0 (C-3′), 148.6 (C-4′), 113.3 (C-5′),121.6 (C-6′), 145.6 (C-7′), 115.0 (C-8′), 97.9 (C-G1), 73.4 (C-G2), 76.4(C-G3), 70.3 (C-G4), 77.5 (C-G5), 61.4 (C-G6).

3-4. Preparation of 6-O-Veratroyl Catalpol from the Extract of P.Longifolium

6.2 g of fraction 4 was subjected to column chromatography. 1.2 g offraction 4-3 was performed to recrystallization in methanol to obtain672.6 mg of 6-O-veratroyl catalpol, i.e., 6-O-3,4-Dimethoxybenzoyl.

6-O-(3,4-dimethoxybenzoyl) catalpol (6-O-veratroyl catalpol)

¹H-NMR (400 MHz, DMSO-d₆) δ: 2.47 (1H, dd, J=8.0, 9.6 Hz, H-9), 2.59(1H, dddd, J=1.6, 4.8, 8.0, 8.0 Hz, H-5), 3.00 (1H, m, H-G4), 3.05 (1H,m, H-G2), 3.14 (1H, m, H-G5), 3.18 (1H, m, H-G3), 3.42, 3.71 (2H, m,H-G6), 3.70 (1H, br s, H-7), 3.72, 3.90 (2H, d, J=13.2 Hz, each, H-10),4.61 (1H, d, J=7.6 Hz, H-G1), 4.97 (1H, dd, J=4.8, 6.0 Hz, H-4), 5.08(1H, d, J=8.8 Hz, H-6), 5.10 (1H, d, J=9.6 Hz, H-1), 6.42 (1H, dd,J=1.6. 6.0 Hz, H-3), 7.09 (1H, d, J=8.4 Hz, H-5′), 7.46 (1H, d, J=2.0Hz, H-2′), 7.64 (1H, dd, J=2.0, 8.4 Hz, H-6′), 3.81, 3.84 (6H, s each,3′,4′-OCH₃).

¹³C-NMR (100 MHz, DMSO-d₆) δ: 92.9 (C-1), 141.1 (C-3), 101.8 (C-4), 35.2(C-5), 79.9 (C-6), 58.2 (C-7), 65.9 (C-8), 41.8 (C-9), 58.4 (C-10),121.3 (C-1′), 111.8 (C-2′), 148.5 (C-3′), 153.2 (C-4′), 111.2 (C-5′),123.5 (C-6′), 165.5 (C-7′), 97.8 (C-G1), 73.4 (C-G2), 76.4 (C-G3), 70.3(C-G4), 77.5 (C-G5), 61.4 (C-G6), 55.6, 55.7 (3′,4′-OCH₃).

3-5. Preparation of Minecoside from the Extract of P. Longifolium

261.0 mg of fraction 4-4 and 288.0 mg of fraction 4-5 were subjected torepeated silica gel column chromatography (chloroform-methanol mixture,methanol 10-20% step gradient) to obtain 52.5 mg of minecoside, i.e.,6-O-3-hydroxy-4-methozycinnamoyl catalpol.

6-O-3-hydroxy-4-methozycinnamoyl catalpol(minecoside)

¹H-NMR (400 MHz, DMSO-d₆) δ: 2.46 (1H, m, H-9), 2.48 (1H, m, H-5), 3.00(1H, m, H-G4), 3.05 (1H, m, H-G2), 3.14 (1H, m, H-G5), 3.18 (1H, m,H-G3), 3.42, 3.70 (2H, m, H-G6), 3.67 (1H, br s, H-7), 3.72, 3.91 (2H,d, J=13.2 Hz, each, H-10), 4.61 (1H, d, J=8.8 Hz, H-G1), 4.94 (1H, dd,J=4.0, 6.0 Hz, H-4), 5.00 (1H, d, J=7.2 Hz, H-6), 5.09 (1H, d, J=9.2 Hz,H-1), 6.42 (1H, dd, J=1.2, 5.6 Hz, H-3), 6.96 (1H, d, J=8.0 Hz, H-5′),7.13 (1H, d, J=2.0 Hz, H-2′), 7.17 (1H, dd, J=2.0, 8.0 Hz, H-6′), 3.82(3H, s, —OCH₃).

¹³C-NMR (100 MHz, DMSO-d₆) δ: 93.0 (C-1), 141.1 (C-3), 101.7 (C-4), 35.1(C-5), 79.3 (C-6), 58.2 (C-7), 65.7 (C-8), 41.8 (C-9), 58.5 (C-10),126.8 (C-1′), 114.5 (C-2′), 146.7 (C-3′), 150.2 (C-4′), 112.0 (C-5′),121.4 (C-6′), 145.7 (C-7′), 114.5 (C-8′), 97.9 (C-G1), 73.4 (C-G2), 76.4(C-G3), 70.3 (C-G4), 77.5 (C-G5), 61.4 (C-G6), 55.6 (4′-OCH₃).

3-6. Preparation of Catalpol from the Extract of P. Longifolium

Verproside was hydrolyzed to yielded catalpol (compound 1) with 0.1N ofKOH. The solution was stirred for 8 hours at room temperature andneutralized with 0.1N of HCL solution. The product was concentrated byrotary evaporator under reduced pressure, subjected to reversed phasesilica gel column (RP18, methanol/water, 1/4, v/v), and yielded 54.0 mgof catalpol.

Catalpol

¹H-NMR (400 MHz, DMSO-d₆) δ: 2.12 (1H, dddd, J=1.6, 4.0, 8.0, 8.0 Hz,H-5), 2.31 (1H, d, J=8.0, 9.6 Hz, H-9), 3.00 (1H, m, H-G4), 3.05 (1H, m,H-G2), 3.11 (1H, m, H-G5), 3.17 (1H, m, H-G3), 3.34 (1H, br s, H-7),3.40, 3.70 (2H, m, H-G6), 3.63, 3.87 (2H, d, J=12.8, each, H-10), 3.76(1H, d, J=8.0 Hz, H-6), 4.59 (1H, d, J=8.0 Hz, H-G1), 4.90 (1H, d, J=9.6Hz, H-1), 5.01 (1H, dd, J=4.6, 6.0 Hz, H-4), 6.36 (1H, dd, J=1.6, 6.0Hz, H-3).

¹³C-NMR (100 MHz, DMSO-d₆) δ: 93.2 (C-1), 140.2 (C-3), 103.3 (C-4), 37.4(C-5), 77.1 (C-6), 60.7 (C-7), 64.8 (C-8), 42.1 (C-9), 58.9 (C-10), 97.8(C-G1), 73.4 (C-G2), 76.4 (C-G3), 70.2 (C-G4), 77.4 (C-G5), 61.3 (C-G6).

Experimental Example 1 MTT Assay

To investigate the cytotoxic effect of inventive extract of P.longifolium extract and the compound isolated therefrom was determinedby (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide(MTT)assay method (Wang Z et al., Biol., Pharm. Bull., 24, pp 159-162, 2001).

Promyelotic HL-60 cells (HL-18103, 5×10⁵ cells/ml) were seeded in96-well plates under NGF-free condition. After 24 hours incubation, thecells were treated with the mixture of samples dissolved in 10 μl ofDMSO and 10 μl of MTT solution (5 mg/ml), and incubated for 4 hoursunder similar condition. 4 hours later, MTT was removed and 100 μl ofDMSO was dropped into each well to dissolve crystals. At 570 nm, UVabsorbance was measured by microplate reader (BIO-RAD, U.S.A.) tocalculate the cell viability.

As shown in Table 1, the result demonstrates that the cell viabilityranges from 98% to 116% in 50 μM, from 95% to 114% in 100 μM. It isconfirmed that an inventive extract or compound the present inventionhas no cell toxicity.

TABLE 1 Effect of compounds isolated from P. longifolium on HL-60 cells.Cell viability (%) Sample 50 μM 100 μM Verposide 105 102 6-O-veratroylcatalpol 116 114 Minecoside 98 95

Experimental Example 2 Airway Hyperresponsiveness (AHR)

The AHR was evaluated by calculation Penh values (enhanced pause) 24hours after the final OVA challenge. The Penh value of the OVA-treatedgroup was significantly higher than that of the PBS control group. Inthe P. longifolium extract +OVA-challenged group, the Penh value wassignificantly reduced compared with that of the OVA-treated group at 30mg/ml methacholine. (table 2) In the verproside+OVA-challenged group,the Penh value was significantly reduced compared with that of theOVA-treated group (P<0.05) A positive control, montelukast (ML), whichhas been widely used as an anti-asthmatic drug, showed a similardecrease of AHR with verproside (FIG. 1).

TABLE 2 Effect of P. longifolium extract on airway hyperresponsiveness(AHR) methacholine Penh value (mg/ml) 0 5 10 30* OVA- 0.66 ± 0.23 1.79 ±0.47 2.75 ± 0.91 4.59 ± 1.07 challenged group OVA + 0.65 ± 0.18 1.33 ±0.53 2.46 ± 0.26 2.85 ± 0.72* P. longifolium (—) (25.7%) (10.5%) (38.0%)extract (% inhibition) *significant difference from OVA-treated group p< 0.05

Experimental Example 3 Effect of P. Longifolium on OVA-InducedEosinophilia in BALF

3-1. Animal Sensitization and Airway Challenge

Specific pathogen-free female BALB/c mice aged 8-10 weeks, which wereroutinely screened serologically for relevant respiratory pathogens,were purchased from ORIENT Co Ltd (Seoul, Korea).

Following treatment: (1) sham-sensitization plus challenge withphosphate-buffered saline (PBS; ipNeb); (2) sensitization plus challengewith OVA (ovalbumin: Sigma A5503; Sigma, St. Louis, Mo.)(ipNeb); (3)sensitization with OVA (i.p.) plus challenge with OVA (Neb) and samples(extract of P. longifolium or montelukast) was performed to Group ofmice (n=5). Briefly, mice were sensitized by intraperitoneal injectionof 20 μg OVA, which was emulsified with 2 mg aluminum hydroxide in 100μl of PBS buffer (pH 7.4) on days 0 and 14. The mice were challengedthrough the airways with OVA (1% in PBS) for 20 min using an ultrasonicnubuilizer (NE-U12; Omron Corp., Tokyo, Japan) on days 28, 29 and 30after the initial sensitization. The mice were sacrificed 48 h after thelast challenge (day 32) to determine the suppression effect of extractof P. longifolium or verproside on the airways of allergic asthma.

3-2. Sample Treatment

The extract of P. longifolium, and verproside were suspended in PBS andadministered intragastrically using a 25-gauge stainless steel bluntfeeding needle 1 h before each challenge, and control animals wereexposed only on the PBS solution. As a positive control, montelukast(MSD Korea Ltd., Seoul, Korea) was treated with the same procedure inthe experiment.

The mice were sacrificed with an overdose of pentobarbital (Sigma P3761)24 h after the last challenge, and a tracheotomy was performed. Afterice-cold 0.5 ml of PBS was instilled into the lungs, bronchoalveolarlavage fluid (BALF) was obtained by aspiration three times (total 1.5ml) via tracheal cannulation (Yamazaki T, J. Jap. Bot., 43, pp 117-24,1968)

3-3. Inflammatory Cell Counts in Bronchoalveolar Lavage Fluid

The total inflammatory cell number was assessed by counting cells in atleast five squares of a hemocytometer after excluding dead cellsconfirmed by staining with trypan blue (Daigle I. et al., Swiss MedWkly, 131, pp 231-7, 2001). 100 μl of BALF was loaded onto a slide andcentrifuged (200×g, 4° C., 10 min) to fix the cells onto the slide usinga cytospine machine (Hanil Science Industrial, Korea). The cells werestained by Diff-Quick® Stain reagents (Sysmex, Cat No. 38721,Switzerland) according to the manufacturer's instructions. Statisticalsignificance was determined by Student's two-tailed t-test forindependent means and the critical level for significance was set atP<0.05.

To evaluate the suppression of verproside on the eosinophilia inOVA-challenged mice, the recruited cells in BALF were counted 48 hoursafter the last challenge. OVA caused a marked influx of leucocytes intothe BALF from a PBS control group. As shown in FIG. 2, the total cellswere counted as 40.5±16.4×10⁴ cells/mouse (P<0.001) compared with thePBS-treated control (2.3±0.6×10⁴ cells/mouse). Eosinophils were found tobe less than 5% of total cells in the PBS-treated mice, however, thesewere increased dramatically to be more than 75% of total leukocytes inthe BALF of OVA-challenged mice. In the verproside-treated mice, thecell migration was significantly attenuated; 79.3±13.1% decrease intotal cells (P<0.005) and 86.2±7.2% in eosinophils (P<0.001) from aOVA-treated control group. A positive control, montelukast (ML), showeda similar suppressive effect of leukocyte influx in BALF as 78.3±12.1%decrease in total cells (P<0.005) and 80.7±11.1% decrease in eosinophils(P<0.005) (FIG. 2). In the treatment of P. longifolium extract +OVA, therecruitments of cells were significantly attenuated also; 66.0±13.2%decrease in total cells and 75.8±7.6% decrease in eosinophils,respectively.

Experimental Example 4 Lung Histology

To estimate the suppressive effect of verproside on the eosinophilia,lung tissues were collected 48 hours after the last challenge. The lungtissue was fixed for 24 h in 10% neutral-buffered formalin. After beingembedded in paraffin, it is sliced into 4-μm thickness of sections andthe tissue was stained with H&E solution (hematoxylin; Sigma MHS-16 andeosin, Sigma HT110-I-32). In the OVA-challenged mice, leukocytes werefound to be infiltrated into the peri-bronchiole and peri-vascularconnective tissue; of these leukocytes, eosinophilia was mainly observed(FIG. 3-II, P<0.005). In the verproside +OVA-challenged mice, theinfiltration of eosinophil-rich leukocytes was significantly attenuatedcompared with the OVA-treated mice (FIG. 3-III, P<0.05). The suppressiveeffect of Montelukast (ML) was shown similar with that of verproside(FIG. 3-IV, P<0.05). In the treatment of P. longifolium extract +OVA,the suppressive effect of leukocyte infiltration was found clearly (FIG.3-V).

In Periodic acid Schiff (PAS) staining, mucus overproduction in theOVA-treated mice was clearly observed as a violet color in the bronchialairways compared with the normal mice. In contrast, mucus was markedlydiminished in the verproside +OVA-challenged mice (FIG. 4). Goblet-cellhyperplasia in the airway epithelium was quantified based on afive-point system: 0, no goblet cells; 1, <25% of the epithelium; 2,25-50% of the epithelium; 3, 50-75% of the epithelium; 4, >75% of theepithelium. For each mouse, five airway sections that were randomlydistributed throughout the left lung were analysed, and their averagescores were calculated. Quantitative analysis of mucus production wasperformed using an image analyzer (Leica Microsystem Imaging solutionLtd.; Cambridge, UK). As shown in FIG. 5, the mucus area was scored as3.60±0.64 in the OVA-treated mice compare with PBS-treated mice (P<0.05)and it was significantly decreased to 1.43±0.23 in the verproside+OVA-treated mice (P<0.05), which was even lower than the positivereference, montelukast (1.53±0.24, P<0.05).

These results demonstrated that verproside reduced eosinophilia andmucus hypersecretion significantly in the airway remodeling process.

Experimental Example 5 Measurement of IgE and Cytokines

Complementary capture and detection antibody pairs for mouse IgEantibodies were purchased from PharMingen (San Diego, Calif.), and theIgE enzyme-linked immunosorbent assay (ELISA) was performed according tothe manufacturer's directions. Duplicate samples in plasma were dilutedto 1:100. IgE levels in each sample were measured from optical densityreadings at 450 nm, and IgE concentrations were calculated from astandard curve that was generated using recombinant IgE (5-2,000 ng/ml).The amount of IL-4 and IL-13 contained in BALF was measured with aspecific mouse ELISA kit (R&D Systems; Minneapolis, Minn.). Thedetection limit of the assays was 250 pg/ml.

As shown in FIG. 6-A and 6-B, The IgE levels were found to be greatlyincreased in the OVA-treated mice: 85.6±17.3 μg/ml (plasma, P<0.05) and59.4±38.4 (BALF, P<0.005) compared with the PBS-treated mice (16.9±23.9μg/ml in plasma, 1.0±0.1 ng/ml in BALF). The IgE levels ofverproside-treated mice were significantly reduced to 40.2±13.2 μg/ml(plasma, P<0.005) and 21.5±11.2 ng/ml (BALF, P<0.05). In the case ofmontelukast, the IgE levels were much lower as 31.4±14.2 μg/ml (plasma,P<0.005) and 3.8±0.7 ng/ml (BALF, P<0.05)

To determine the effect of verproside on cytokine release in theOVA-induced asthmatic mice, the levels of cytokines (IL-4 and IL-13) inBALF were measured using ELISA 48 hours after the last challenge. OVAchallenge induced a significant elevation of the cytokines to 14.1±6.1pg/ml (IL-4) and 178.5±96.4 pg/ml (IL-13) in the BALF compared with thecontrol (IL-4, 0.1±0.5 pg/ml; IL-13, 0.1±1.0 pg/ml). In theverproside-treated group, the cytokines were significantly suppressed;64.5±27.7% decrease in IL-4 (P<0.05) and 74.9±15.5% in IL-13(P<0.005)from a OVA-challenged group. Montelukast also showed a significantreduction in both of IL-4 (69.5±22.0% decrease, P<0.05) and IL-13(84.5±8.2% decrease, P<0.05) from the control. These results demonstratethat verproside reduced the IL-4 and IL-13concentrations in the BALF ofthe asthmatic model as montelukast did (FIG. 6-C and 6-D).

Hereinafter, the formulating methods and kinds of excipients will bedescribed, but the present invention is not limited to them. Therepresentative preparation examples were described as follows.

Preparation of Injection

Dried powder of Example 1 or verproside 100 mg  Sodium metabisulfite 3.0mg Methyl paraben 0.8 mg Propyl paraben 0.1 mg Distilled water forinjection optimum amountInjection preparation was prepared by dissolving active component,controlling pH to about 7.5 and then filling all the components in 2 mLample and sterilizing by conventional injection preparation method.Preparation of Powder

Dried powder of Example 1 or verproside 500 mg Corn Starch 100 mgLactose 100 mg Talc  10 mgPowder preparation was prepared by mixing above components and fillingsealed package.Preparation of Tablet

Dried powder of Example 1 or verproside 200 mg Corn Starch 100 mgLactose 100 mg Magnesium stearate optimum amountTablet preparation was prepared by mixing above components andentabletting.Preparation of Capsule

Dried powder of Example 1 or verproside 100 mg  Lactose 50 mg Cornstarch 50 mg Talc  2 mg Magnesium stearate optimum amountTablet preparation was prepared by mixing above components and fillinggelatin capsule by conventional gelatin preparation method.Preparation of Liquid

Dried powder of Example 1 or verproside 1000 mg Sugar 20 gPolysaccharide 20 g Lemon flavor 20 gLiquid preparation was prepared by dissolving active component, and thenfilling all the components in 1000 mL ample and sterilizing byconventional liquid preparation method.Preparation of Health Food

Dried powder of Example 1 or verproside 1000 mg Vitamin mixture optimumamount Vitamin A acetate 70 μg Vitamin E 1.0 mg Vitamin B₁ 0.13 mgVitamin B₂ 0.15 mg Vitamin B6 0.5 mg Vitamin B12 0.2 μg Vitamin C 10 mgBiotin 10 μg Amide nicotinic acid 1.7 mg Folic acid 50 μg Calciumpantothenic acid 0.5 mg Mineral mixture optimum amount Ferrous sulfate1.75 mg Zinc oxide 0.82 mg Magnesium carbonate 25.3 mg Monopotassiumphosphate 15 mg Dicalcium phosphate 55 mg Potassium citrate 90 mgCalcium carbonate 100 mg Magnesium chloride 24.8 mgThe above mentioned vitamin and mineral mixture may be varied in manyways. Such variations are not to be regarded as a departure from thespirit and scope of the present invention.Preparation of Health Beverage

Dried powder of Example 1 or verproside 1000 mg Citric acid 1000 mgOligosaccharide 100 g Apricot concentration 2 g Taurine 1 g Distilledwater 900 mLHealth beverage preparation was prepared by dissolving active component,mixing, stirred at 85° C. for 1 hour, filtered and then filling all thecomponents in 1000 mL ample and sterilizing by conventional healthbeverage preparation method.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the present invention, and allsuch modifications as would be obvious to one skilled in the art areintended to be included within the scope of the following claims.

INDUSTRIAL APPLICABILITY

As described in the present invention, the extract of P. longifolium andthe catalpol derivatives isolated therefrom show the suppression ofelevated IgE, IL-4 and IL-13 levels and eosinophilia in plasma and BALD,and mucus overproduction in the lung tissues using by OVA-inducedasthmatic mouse model. Therefore, it can be used as the therapeutics orfunctional health food for treating and preventing inflammatory,allergic and asthmatic disease.

We claim:
 1. A method of treating inflammation and allergy, comprisingadministering a composition consisting of an extract ofPseudolysimachion genus plant as an active ingredient and apharmaceutically acceptable carrier to a subject in need thereof.
 2. Themethod of claim 1, wherein the Pseudolysimachion genus plant is selectedfrom the group consisting of P. longifolium, P. ovatum, P. kiusianum, P.kiusianum var. diamanticum, P. kiusianum var. villosum, P. dahuricum, P.pyrethrinum, P. linarifolium, P. linarifolium var. villosulum, P.rotundum var. subintegrum, P. rotundum var. coreanum, and P. insulare.3. The method of claim 1, wherein the extract is prepared by extractingthe Pseudolysimachion genus plant with a solvent selected from the groupconsisting of water, C₁-C₄ alcohols, acetone, ethyl acetate, chloroform,dichloromethane and the mixtures thereof.
 4. The method of claim 1,wherein the extract comprises catalpol derivatives represented bygeneral formula (I) or pharmaceutically acceptable salt thereof:

wherein, R is selected from the group consisting of hydrogen,3,4-dihydroxybenzoyl, 3-hydroxy -4-methozybenzoyl, 4-hydroxybenzoyl,3,4-dimethoxybenzoyl and cinnamoyl group substituted with 3,4-dihydroxy,3-hydroxy-4-methoxy, C₁-C₃ lower alkyl group or C₁-C₃ lower alkoxygroup.